The zeta isozyme of protein kinase C (PKCzeta) has a widespread distribution. It is atypical in that it is not regulated by Ca2+ or phorbol ester/diacylglycerl, and its cellular regulation and functions are unknown. Recently, we observed that it was activated by phosphatidylinositol 3,4,5-triphosphate (PIP/3). This is a product of phosphatidylinositol 3-kinase (PI 3-kinase), which is activated in many cells by insulin, growth factors, hormones and oncogenes. Like PKCzeta, its cellular role remains unclear. In this proposal, fibroblasts and other cell types will be used to identify agonists that activate PKCzeta by studying its translocation, autophosphorylation and the phosphorylation of its specific substrates. The role of PIP/3 in this activation will be examined by performing correlative measurements of PIP/3 and using agonists that selectively alter the levels of different regulatory lipids. Activation of the enzyme will be studied in cells expressing mutant PDGF receptors that differ in their coupling to PI 3-kinase, and in cells treated with the PI 3-kinase inhibitor wortmannin and with rapamycin. Other potential lipid regulators of the enzyme will be examined by measuring their levels and using exogenous phospholipases to generate them. Cellular substrates of PKCzeta will be identified by several approaches including phosphorylation of cell extracts and candidate substrates in vitro, a gel overlay approach, and the yeast two-hybrid system, which will be used to screen a kidney cDNA library. The role of PKCzeta and of fusion proteins containing segments of the PKCzeta regulatory domain. Cells expressing a dominant negative PKCzeta mutant will also be studied, as will be cells depleted PKCzeta by antisense oligonucleotide and RNA approaches. In all these cells with inhibited or depleted PKCzeta, changes in DNA synthesis and growth in response to selected agonists will be measured and these will be correlated with changes in the activity of p70/S6K and any protein kinase that is shown to be a substrate of PKCzeta. The ultimate aim will be to define the signalling pathway(s) involving PKCzeta that controls growth. The proposed studies should define the physiological role and regulation of PKCzeta and determine the roles of PIP/3 and PKCzeta in the mechanisms of action of insulin and growth factors. They should therefore contribute to understanding of diabetes mellitus and of factors involved in normal and abnormal growth of cells.